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I. Site-directed mutagenesis PCR and DpnI digestion (Video)
1. Go to Additional Protocols, Designing Mutation Primers, to see how to design mutation primers. Use the Mutating Primer Master list on the computer to determine which enzyme mix to use: Q5 Master Mix or PfuUltra Master Mix. Find the PCR mix (Q5 or PfuUltra) and the template DNA in the PCR box (Box 12, Freezer C). Forward and reverse primers are in the primer box for the appropriate protein (Box 9 for TAAR1, Box 10 for CFTR, Box 9B for B2AR and some TAAR1, Freezer C). Grab the smaller primer tubes.
2. Locate the PCR mix you are using in the tables below. In a PCR tube, mix the reagents in the order indicated on the table.
3. Before cycling, ensure the PCR tubes do not have any air bubbles. If there are air bubbles present, tap the tubes on the table, and/or centrifuge them in the mini centrifuge. You may also flick them with your finger followed by spinning.
When you order primers from Genewiz or Sigma, the typical amount we get is around 25 nmol (check on the tube). If you resuspend this in 250 µL of water, the concentration is 100 µM. In order to have the 10 µM working solution, you need to do a further 10-fold dilution.
*PCR machine is on the first floor: 112C. Blue key needed.
There is a smaller thermocycler in the lab (check the back). Video
Cycling Conditions
*Annealing temperatures may vary depending on the primers being used. Set it for 2-3°C lower than the lowest Tm for the primer(s) you're using.
**Extension time may need to be adjusted depending on the size of the plasmid. Assume a rate of 1 kb/minute to calculate a new time.
4. At the end of the reaction, place the tubes in the PCR Rack (Freezer A) if you’re not doing the next steps right away.
5. If you continue with the protocol, take 2 µL out of every PCR tube to prepare samples for gel electrophoresis analysis. If the reactions succeeded, draw a check mark on the side of the tube and continue with the next step. If the reaction failed, discard the tube in the trash and rethink the PCR strategy.
6. For the successful reactions, add 1 µL of DpnI (Enzyme Rack, Freezer A) to the remaining PCR product and digest at 37°C overnight. Write DpnI on the side of the tube. If in a rush, you may add 1 µL of FD DpnI and digest at 37°C for two hours.
NOTE: If PfuUltra Master Mix was used, skip to Transformation. If Q5 Master Mix was used, the linear PCR product must be ligated into circular DNA before transformation. See below.
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